[0001] The present invention regards an implanting device whose surface combines antibacterial
activity to prevent periprosthetic infections and improved osteointegration capacity.
Background of the invention
[0002] In orthopaedics, periprosthetic infection (PPI) is a devastating consequence of the
insertion of implants. Currently the incidence of PPI ranges from 1 to 5%, with even
greater values for high risk patients and for those that have suffered a trauma. Furthermore,
about 73% of the operation reviews are carried out following a peri-implant bacterial
infection. The social and economical cost of these interventions is considerably high.
The etiology of PPI is complex and it depends on the ability of the bacterial species
to elude the response of the host tissue, usually through the formation of a biofilm.
Actually, once inserted, the implant device is coated by serum proteins. This process
is followed by interactions with the cell species and the regeneration or reparation
of the tissue in cases with positive course. In presence of bacterial species, the
surface of the implant may be subjected to the bacterial adhesion and the formation
of a biofilm. In particular, within the biofilm the bacteria are protected by the
immune-surveillance system and by the effect of systemic antibiotics. In this manner,
the colonization may propagate, with the harmful consequences known in the field of
periprosthetic infection. A series of systems for the local release of antibiotics,
both from cements and micrometric layers of biodegradable polymers deposited on the
surface of the devices, were developed with the aim of fighting PPI. However, upon
completing release, the porous systems of this type may serve as a protected site
for the bacterial adhesion and the formation of biofilms.
[0003] Articles of literature have been recently presented in which an antibiotic, vancomycin,
was covalently bonded to the surface of implant devices made of titanium and conducted
bactericidal activity against bacterial species belonging to the Staphylococci genre
(
Chemistry & Biology, Vol. 12, 1041-1048, 2005, Vancomycin Covalently Bonded to Titanium Beads Kills Staphylococcus aureus;
Journal Orthopedic Research, 25, 858-866, 2007, Vancomycin Covalently Bonded to Titanium Alloy Prevents Bacterial Colonization).
[0004] The immobilisation of vancomycin on the surface of implant devices or local release
thereof are, for these applications, processes of great interest. Vancomycin constitutes
a potent drug for treating Gram-positive bacterial infections, which are considerably
the most common cause of periprosthetic infections. The action mechanism of vancomycin
provides for the block of the synthesis of the layer of peptidoglycans of the gram-positive
bacteria cell walls by means of L-Lys-D-Ala-D-Ala terminal bond of the nascent peptidoglycan.
In this manner, vancomycin prevents the crosslinking which is required for the osmotic
stability. The concept of firmly bonding vancomycin, per se water soluble, to the
surface of the implant devices overcomes the vision of the simple release system.
Actually, in this case there is a high local concentration of a drug firmly bonded
to the interface between the implant device and the external environment. This stable
pharmacological barrier prevents the formation of bacterial colonies on the surface
of the implant device, thus preventing the occurrence of PPI. As described by
Chapiro and collaborators in the article Selfprotective Smart Orthopedic Implants,
Expert Rev. Med. Devices, 2007 Jan;4(1):55-64, systems of this type can lead to a new generation of implant devices, which are
self-protected against risks of bacterial infection due to their surface properties.
[0005] However, the process of bonding vancomycin to the surface of the device made of titanium
described in these articles comprises different steps, which are quite complex from
a practice point of view and not easily adaptable to industrial production, which
makes it poorly suitable for devices of a given dimension and complex geometry.
[0008] Obviously, the provision, under conditions compatible with a productive context,
of implant prosthetic devices capable of exploiting the pharmacological action of
vancomycin would constitute a considerable step forward in the sector, with considerable
scientific, social and economical implications. Ideally, the process could accompany
the immobilization/release of vancomycin to provide other considerable surface properties,
such as the increase of the osteointegration speed. Actually, the rapid regeneration
of the bone tissue, with ensuing occupation of the available surface of the implanted
device, reduces the probabilities of surface colonization by bacterial cells, completing
the antibacterial protective effect due to vancomycin. This concept would allow the
actual provision of devices with multifunctional surfaces, i.e. surfaces that perform,
besides the obvious function of supporting the tissue components, also for example:
- the function of stimulating the regeneration/reparation of the tissue
- the function of antibacterial protection.
[0009] Studies carried out by the present inventor have now revealed the possibility of
practically implementing the previously mentioned concepts as described hereinafter.
Summary of the invention
[0010] A first scope of the present invention is to provide an implantable device to be
implanted in a human or animal body, wherein at least one part of the surface of said
device is coated with a complex of hyaluronic acid and glycopeptide antibiotic and
wherein said hyaluronic acid has an average molecular weight comprised between 400.000
and 1.000.000 Da.
[0011] A second scope of the present invention is to provide a collagen gel comprising the
hyaluronic acid complex with a glycopeptide antibiotic, as such or lyophilized, wherein
said hyaluronic acid has an average molecular weight comprised between 400.000 and
1.000.000 Da.
[0012] A third scope of the present invention is to provide a method for obtaining an implantable
device according to the first embodiment or a gel according to the second embodiment,
comprising the step of forming said hyaluronic acid complex with said glycopeptide
antibiotic by making said hyaluronic acid come into contact with a solution of said
glycopeptide antibiotic, in the presence of a condensing or cross-linking agent.
[0013] The present invention regards the process for obtaining an implant device in the
human or animal body capable of combining the immobilization of an antibiotic, in
particular vancomycin, in a simple and efficient manner with the stimulation of the
osteogenic cells and ensuing increase of the osteointegration speed. The rapid formation
of bone tissue, with relative occupation of the available surface of the implanted
device, reduces the probabilities of surface colonization by bacterial cells and thus
constitutes a synergic effect with the antibiotic effect of vancomycin. According
to the process subject of the invention, devices with multifunctional surfaces, i.e.
surfaces that exert, besides the obvious function of supporting the tissue components,
also the mentioned functions of stimulating the regeneration/reparation of the tissue
and antibacterial protection, are thus provided for.
[0014] The present invention is based on the surprising observation that vancomycin, a water
soluble compound, if present in an aqueous solution with the molecule of hyaluronic
acid in presence of cross-linking/condensing agents, forms a compound/precipitate
with said hyaluronic acid. From such observation, a process has been developed which
replicates these events on the surface of implant devices. The process provides for,
at the beginning, the bond of the molecule of hyaluronic acid to a suitably functionalised
surface (with methods known in literature); then, the surface coated with hyaluronic
acid is incubated in an aqueous solution of vancomycin in presence of cross-linking
agents in suitable concentration. Surprisingly, there is gradually observed on the
surface the formation of hyaluronic acid/vancomycin precipitates, observable also
macroscopically due to the gradual formation of an "opaque" layer which homogeneously
coats the entire surface, an event that does not occur if the surface was not previously
coated with hyaluronic acid.
[0015] Without being restricted to a particular theory, it is deemed that this behaviour
is at least partly due to the ionic interaction between the negative charges present
in the molecule of hyaluronic acid and the positively charged amino groups of vancomycin.
However, given that the formation of said opaque layer only occurs in presence of
given cross-linking agents, the interaction could also be due to different physical
and chemical factors, unexpectedly related to the molecular structure of the hyaluronic
acid.
[0016] For the sake of brevity, in the present patent application such precipitates or compounds
of a glycopeptide antibiotic, in particular vancomycin, and hyaluronic acid shall
be defined "hyaluronic acid/glycopeptide antibiotic complexes" or specifically "hyaluronic
acid/vancomycin complexes", without implying that the bond typical of a complex is
necessarily formed.
[0017] Furthermore, it was surprisingly discovered that the use of different molecular weights
of hyaluronic acid (HA) considerably influences the formation of said HA /vancomycin
precipitate: in this case, it was observed that coating a surface with HA with low
molecular weights (comprised between 5000 Da and 80000 Da) is not efficient at "capturing",
in the subsequent treatment step, an amount of vancomycin molecules equivalent to
that captured when the surface is coated with hyaluronic acid with higher molecular
weight. Furthermore, the maximum yield in the capturing process does not increase
as the weight of the hyaluronic acid increases, but it reveals a "peak" or bell development.
[0018] Furthermore, the use of other polysaccharides, among which heparin, chondroitin sulphate,
chitosan, alginic acid, pectins, has the effect of reducing the amount of functional
vancomycin which is bonded on the surface (in some cases reducing stability thereof,
in others the pharmacological activity).
[0019] Surprisingly, the compound/precipitate incorporated in the surface layer is released
if exposed to physiological aqueous environments, preserving the pharmacological action
of vancomycin. The total release of vancomycin is not immediate, as it would be expected
due to the high solubility of vancomycin in aqueous solutions, but the effect remains
for several weeks, offering an extended interfacial antibiotic coating.
[0020] Hyaluronic acid is deposited on the surface of the device to be coated using different
methods that allow pre-treating the surface to be coated with allylamine plasma or
polyethyleneimine aqueous solution, with amino groups capable of allowing covalent
bonding with the carboxyl groups of the hyaluronic acid.
[0021] According to a preferred embodiment, the surface to be treated can be coated with
a single layer of collagen molecules, preferably in fibrillated forms: the amino groups
of the amino acid residues of the collagen molecule are capable of bonding the carboxyl
groups of hyaluronic acid. The functionalization of the surfaces with collagen also
allows increasing the "osteoinductive" properties of the surface: actually, it was
observed that such modification is capable of directing the differentiation of human
mesenchymal cells towards the osteogenic line.
[0022] The previously described process is versatile and it is not limited by the type of
the device. It can thus be applied both on metal devices such as screws made of titanium
for fixing fractures, for prosthesis, on polymeric devices, both biodegradable and
permanent, and on ceramic devices, for example hydroxyapatite or other forms of calcium
phosphates, even in form of particulate like powders and granules as used for void
bone fillers.
[0023] The process of the invention may be applied on shaped bodies based on natural materials
such as sponges or scaffolds based on collagen or natural polymers or ceramic materials.
It can also be applied to suspensions of nanostructured organic material, such as
collagen fibrils with diameter smaller than a micron.
[0024] In case of application to collagen suspensions or nanostructured materials in suspension,
the interaction subject of this invention leads to the formation of a gel. This gel
- bas.ed on collagen and containing vancomycin and hyaluronic acid - can be used,
as it is or in lyophilized forms and reconstituted when using, as filling material
in the site of fixation an implantable device, performing both the typical action
of osteointegration of collagen and hyaluronic acid and the antibacterial activity
of the antibiotic also in the region surrounding the implant.
[0026] Thus, the previously described methods allow providing implant devices that do not
only have optimal and improved osteointegration characteristics but they also provide
the self-protective and bactericidal characteristics of vancomycin, practically translating
the concept of multifunctional surface. Furthermore, the method of the present invention
allows extending the multifunctionality of the surface, exemplified by the simultaneous
presence of the bioactive chemical composition with osteogenic action (hyaluronic
acid bond) and pharmacological action (release of vancomycin), adding the possibility
of controlling the surface topography through roughening processes. Regarding this,
it should be borne in mind that in the field of dental implants the osteointegration
process is facilitated by the action of the surface topography on the osteogenic cells,
as exemplified by the concept of "
Modulation of osteogenesis via implant surface design", described by Boyan, B.D.,
Schwartz, Z., , in: , Davies J. E. editor. Bone Engineering, Toronto, em squared,
232-239, 2000. This concept has been so successful to a point that dental implants with smooth
surface are no longer available in the market all having instead a rough surface topography.
In orthopaedics, for example in the fixation of fractures by means of screws made
of titanium, this concept could not be exploited due to the fear of offering, by roughening
the surface, surfaces that can be exploited for bacterial adhesion and colonization.
The present invention instead allows providing a conformal hyaluronic acid surface
osteogenic layer (i.e. which does not alter the topography of the surface, adapting
thereto), which can also exploit the roughening of the surface topography in that
the release of vancomycin prevents the bacterial colonization of the unevenness and
roughness of the surface.
Description of the invention
[0027] The present invention regards in particular an implanting device in the human or
animal body, in which at least one part of the surface of said device is coated with
a "hyaluronic acid compound with a glycopeptide antibiotic.
[0028] In an embodiment, the glycopeptide antibiotic is vancomycin.
[0029] The idea is to bond a layer of hyaluronic acid to the surfaces of interest (through
the methods known in the art), then incubate such surfaces with a solution of vancomycin
in presence of condensing agents, in particular N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC), present simultaneously: an "opaque" layer, indicating the interaction
between the previously bonded hyaluronic acid and vancomycin is formed on the surface
after a few hours. This opaque layer is not formed in the absence of hyaluronic acid
bonded to the surface, functionalised or non-functionalised, and it is not formed
in absence of NHS/EDC. From now henceforth, reference shall be made to "hyaluronic
acid complexes with vancomycin", where the term "complex" comprises any type of chemical/physical
or simply physical interaction between hyaluronic acid and vancomycin, including hydrogen
bond interactions, and/or Van der Waals interactions and/or electrostatic interactions
etc, which is obtained through the precipitation of vancomycin or coprecipitation
of hyaluronic acid and vancomycin in presence of cross-linking or condensing agents
and in particular conditions (pH and concentration of the solutions).
[0030] In an embodiment, the entire surface of the implanting device is coated with said
hyaluronic acid/glycopeptide antibiotic complex bonded to the surface having a. Alternatively,
part of the device is coated with hyaluronic acid, and the formation of the complexes
with vancomycin only occurs in this area.
[0031] The implanting device according to the invention can be made of metal (for example,
steel, titanium or alloys thereof with other metals) or made of plastic material (such
as for example polystyrene) compatible for applications on the human or animal body,
or made of ceramic material, both in form of the device and granulate/powder.
[0032] In an embodiment, the device consists of a dental implant screw, preferably made
of titanium or alloys thereof, possibly of the transmucosal type, or a screw, preferably
made of titanium or alloys thereof, for spinal or skeletal fixation, or for fracture
fixation, or an intervertebral disc, preferably made of titanium, alloys thereof or
cobalt-chromium alloys or made of metal alloys commonly used for these applications.
The surface of the device made of titanium can be smooth (commonly referred to as
"machined") or preferably roughened according to methods known in the art, in particular
through sand blasting, using alumina, titanium oxides or other sand blasting agents,
roughening treatment using acids, or electrochemical roughening treatment.
[0033] In an embodiment, the process of immobilizing the hyaluronic acid complex with the
glycopeptide antibiotic on an implantable device according to the invention provides
for the introduction of functional amino groups on the surface of the device. Hyaluronic
acid is bonded covalently to the surface amino groups, through the methods known in
the art, and exerts the vancomycin "capturing" action thereof with formation of the
complexes in presence of EDC and NHS in the next step. The average molecular weight
of hyaluronic acid is comprised between 400,000 and 1 million Da. The bond of hyaluronic
acid to the amined surface may occur through the methods known in the art, for example
by using EDC/NHS, or by treating hyaluronic acid with periodate acid and subsequent
reductive amination, or as described in
WO2006/038056 A1. The layer thus obtained may have a thickness comprised between 0.1 and 1000 nanometres,
preferably between 1 and 20 nm.
[0034] The amino groups may be deposited on the surface of the implantable device according
to the methods widely known in the sector. The technique which provides for the introduction
of the substrate having functional amino groups on the surface of the implantable
device through plasma deposition of molecules carrying the amino groups is particularly
advantageous. Typical examples of molecules used for this purpose are allylamine,
alkylamines such as hexylamine or heptylamine and, generally, organic molecules having
amino functions which exhibit the required volatility characteristics in the plasma
state. The plasma deposition of amine occurs in the following conditions: pressure
comprised between 80 and 300 mTorrs, discharge power comprised between 5 and 200 W,
deposition time between 1 ms and 300 s. Plasma deposition may also occur in pulsed
plasma conditions, with active and inactive plasma cycles comprised between 1 and
100 ms, to minimize the molecular fragmentation and maintain the highest density possible
of the amino groups. The plasma deposition treatment of amines may be preceded by
other plasma treatments, for example using air or oxygen plasma for cleaning the surface
and increasing adhesion with the substrate.
[0035] A further method for coating the implantable device with a substrate containing amino
groups consists in adsorbing polyethyleneimine (PEI) on the device surface, for example
from a 0.2% aqueous solution, for 2 hours, at ambient temperature. Also the use of
silanes, for example 3-aminopropyltriethoxysilane (APTES), or analogous compounds,
falls within the common methods of surface functionalization.
[0036] It is also possible to functionalise the surface of the implantable device with carboxyl
groups through plasma deposition of acrylic/methacrylic acid.
[0037] Furthermore, it is possible to functionalise the surface with natural molecules,
possibly bioactive, which are irreversibly adsorbed on the surface and have amino/carboxylic
groups suitable for the subsequent bond of hyaluronic acid and complexes thereof with
vancomycin. Typical examples falling within this category are collagen and other extracellular
matrix protein molecules, such as laminin, fibronectin, vitronectin. According to
this embodiment, the implanting device according to the invention comprises a first
layer of adsorbed collagen, in a monomer or fibrillated form, to which hyaluronic
acid is bonded, according to the described process, preferably through EDC/NHS, or
through reductive amination. This is followed by the formation of the hyaluronic acid/vancomycin
complexes, through exposure to vancomycin and cross-linking or condensing agents solutions.
The functionalization with collagen is preferably performed with a 0.3% collagen solution
in 10mM acetic acid and an equal volume of phosphate buffer at 37°C for 8 hours.
[0038] The previously described method may also be performed on collagen in suspension,
leading to the formation - in the aqueous medium - of a precipitate of complex of
fibrillated collagen, hyaluronic acid and glycopeptide antibiotic (such as vancomycin),
which can be separated, washed and partly dried to obtain a gel. This gel based on
collagen and containing said glycopeptide antibiotic and hyaluronic acid can be used,
as it is or in lyophilized forms and reconstructed when using, as filling material
in the site of fixation of said implantable device, performing both the typical osteointegration
action of collagen and hyaluronic acid as well as the antibacterial activity of the
antibiotic also in the region surrounding the implant.
[0039] A further object of the invention is a kit comprising an implanting device on the
human or animal body, in which at least one part of the surface of said device is
coated with a hyaluronic acid complex with a glycopeptide antibiotic, and a fibrillated
collagen gel with said hyaluronic acid/glycopeptide antibiotic complex.
[0040] The following examples describe the invention.
Example 1. Hyaluronic acid-vancomycin interaction.
[0041] This example shows that there is a surprising interaction between vancomycin and
hyaluronic acid.
[0042] A 0.1% (w/v) solution of hyaluronic acid HW (molecular weight 800 kDa, Lifecore)
in milliQ water and a 0.5% vancomycin solution in milliQ water is prepared. The hyaluronic
acid solution is gradually added to the latter solution and a gradual increase of
the turbidity of the solution up to the formation of particles in suspension is observed
(sign of strong interaction between HA and vancomycin). If two condensing agents,
N-hydroxysuccinimide and subsequently ethyl-carbodiimide are added to the mixture,
the solution turns instantly limpid. However, after 15 hours, the mixture becomes
very turbid, with the presence of precipitates in suspension: probably the condensing
agents are capable of bonding the amino groups of vancomycin to the carboxyl groups
of the hyaluronic acid. Such precipitates were collected, washed with milliQ water
and subsequently analysed under IR: figure 1 shows the spectra relative to vancomycin
(dots), hyaluronic acid (dashes) and the precipitate formed through the described
method (continuous). In this spectrum there are signals of vancomycin and signals
of hyaluronic acid, confirming interaction between the two molecules.
Example 2. Hyaluronic acid-vancomycin interaction.
[0043] This example shows the fundamental role of condensing agents in the interaction between
vancomycin and hyaluronic acid.
[0044] The previous experiment is repeated using hyaluronic acid and vancomycin solutions
at very low concentrations (up to 10 times lower): in these cases, the addition of
hyaluronic acid to a vancomycin solution does not cause an immediate turbidity of
the mixture, which remains limpid. However, by adding the same amounts of condensing
agents used in the previous experiment and leaving in incubation for about 15 hours,
the solution turns turbid (even though at a lower degree with respect to the large
precipitates formed in the previous experiment), proving that the HA-vancomycin interaction
is somehow "stimulated" by the presence of condensing agents.
[0045] Solutions with high concentration of vancomycin in water (1%) and HA in water (0.5%)
were prepared with the aim of observing possible differences between the HA-vancomycin
interaction that occurs in presence or in absence of condensing agents. Such solutions
were mixed and the precipitate immediately formed was collected, washed and analysed
under IR. The spectrum relative to this precipitate was then compared with that relative
to the precipitate that is formed in presence of condensing agents.
[0046] Figure 2 shows - through a dotted and solid line - the spectra of HA and vancomycin
respectively, while the long dash line shows the spectrum of the HA-vancomycin precipitate
in presence of condensing agents and the short dash line shows the spectrum of the
precipitate in absence of condensing agents.
[0047] There are two important signals present in the spectrum in long dash line and not
in the short dash line: the first is at 1550 cm-1, which could be the signal of the
secondary amide (bending NH2 and stretching CN); the second is at 840 cm-1 and it
could be the signal of aliphatic amines and the bending OCN. These two signals, solely
present in the spectrum of the precipitate formed in presence of condensing agents
(but not actually belonging to the condensing agents), could indicate that bonds different
from those deriving from simple electrostatic interaction, such as covalent bonds,
specific interactions or other interactions formed in such precipitate.
[0048] Potential existence of a strong interaction between hyaluronic acid and vancomycin
has paved the way towards new surface modification experiments. Such experiments consist
in covalently bonding hyaluronic acid to a suitably functionalized surface, then incubating
the abovementioned surface in a solution of vancomycin in presence of condensing agents,
so as to directly provide the same reaction observed in the solution on the surface.
Example 3. HA-vancomycin interaction on a surface
[0049] This example shows that the process observed in solution also occurs directly on
the surface of a material.
[0050] Polystyrene surfaces were prepared as follows:
- air plasma treatment for 20 seconds
- incubation with Polyethyleneimine solution (PEI) 0.5% in water for 2 hours
- washing with milliQ water (3 times)
- incubation of the polystyrene surfaces O.N. with hyaluronic acid solution 800 kDa
0.2% (Lifecore) in milliQ water, in presence of 5 mg/cc NHS and 7.5 mg/cc EDC.
- washing with milliQ water (2 times)
- incubation with 0.7% vancomycin solution in water overnight, in presence of 5 mg/cc
NHS and 7.5 mg/cc EDC.
- Washing with milliQ water (3 times)
[0051] At the end of the treatment on the polystyrene surfaces thus prepared a very homogeneous
opaque thin layer, derived from the occurred interaction between hyaluronic acid bonded
on the surface in the first reaction step and the vancomycin added in the second reaction
step is observed. Polystyrene surfaces treated in the same manner but in absence of
NHS and EDC during the second incubation in vancomycin solution, are perfectly transparent
hence confirming the importance of the condensing agents.
Example 4 - Process for coating an implant screw made of titanium with hyaluronic
acid and vancomycin
[0052] Some implant screws made of titanium, with a length of 13 mm and width of 4 mm, are
treated as follows:
sample 1: no treatment (Ti sample)
sample 2: surface functionalization using PEI, and HA bonding like in example 3 (Ti-HA
Sample).
sample 3: a sample treated like in point 2 was subsequently subjected to incubation
with a 0.7% vancomycin solution in water overnight, in presence of 5 mg/cc NHS and
7.5 mg/cc EDC and subsequent washing with milliQ water (Ti-HA-VXL sample)
[0053] The samples thus obtained were subsequently subjected to XPS (X-ray Photoelectron
Spectroscopy) analysis to evaluate the chemical composition of the surface. A sample
of vancomycin, obtained by leaving a solution of vancomycin in milliQ water to evaporate
on a plastic substrate was analysed therealong as reference. The following results
were obtained, expressed in percentage of atoms and reminding that the XPS analysis
does not measure the presence of hydrogen atoms:
Sample |
C |
O |
N |
Ti |
Cl |
Vancomycin |
77.8 |
15.9 |
4.6 |
|
1.7 |
Ti |
34.8 |
46.4 |
0.4 |
18.4 |
|
Ti-HA |
69.0 |
24.4 |
6.3 |
0.3 |
|
Ti-HA-VXL |
69.5 |
23.2 |
5.8 |
0.6 |
0.9 |
[0054] The vancomycin molecule is characterised, for analytical purposes regarding this
evaluation, by the presence of the Cl hetero-element (two atoms in a compound also
comprising O, C and N with molecular weight of about 1450 Da). The presence of Cl,
at an atomic percentage lower than 2%, is actually shown by the XPS analysis, as indicated
in the table, in the line indicating the analysis of Vancomycin. The surfaces of the
Ti screw and of the Ti-HA screw are characterised by composition values in line with
the expectations, as observable from the literature of the sector.
[0055] The composition of the surface of the Ti-HA-VXL sample is different from that of
Ti-HA due to the presence of Cl, hence the introduction of the Vancomycin molecule
on the surface.
Example 5 - Example of the importance of the molecular weight of hyaluronic acid on
the release of vancomycin from surfaces modified through the present process
[0056] Modified polystyrene surfaces with different molecular weights of HA and then with
the vancomycin solution in presence of NHS and EDC were prepared to observe the specificity
of the HA-vancomycin reaction. HA LW (10 kDa), HA MW (about 70 kDa), HA HW (880 kDa)
and HA HHW (about 2000 kDa) were used. Thus, the surfaces were first functionalised
with polyethyleneimine (0.5% solution in water for 2 hours); then incubation was conducted
overnight with the HA solutions with different molecular weight in presence of NHS
and EDC. Washing was subsequently carried out in water and then the surfaces were
incubated overnight with a 0.8% vancomycin solution in water in presence of 5 mg/ml
NHS and 7.5 mg/ml of EDC.
[0057] The opacity of the surface was observed solely with the HA HW weight, even though
for HA MW and HA HHW the wells did not appear perfectly transparent: thus, there is
a certain specificity, even though only dimensional, in the interaction between vancomycin
and HA HW.
[0058] At the end of the washing, the surfaces were incubated in PBS to conduct the analysis
of vancomycin release over time through HPLC. The chart in figure 3 shows the amount
of vancomycin released by the different surfaces over time (one month): it can be
observed that the surface modified with HA HW releases a greater amount of antibiotic
with respect to those modified with the other molecular weights.
Example 6 - Influence of the functionalization process on the amount of vancomycin
released by surfaces modified through the present process
[0059] The treatment of two 6-wells of polystyrene with HA + VXL (VXL = Vancomycin and condensing
agents EDC-NHS), but functionalizing the surfaces with PEI (polyethyleneimine) or
with fibrillated collagen was planned to verify whether functionalization with fibrillated
collagen causes some changes in the rate of releasing vancomycin from the polystyrene
surfaces. The 6-wells were plasma treated like in the previous example, then the wells
were incubated with a solution of PEI 0.5% in water for 2 hours or with a 0.3% collagen
solution in 10mM acetic acid and an equal volume of PBS a 37°C for 8 hours.
[0060] Before the step of functionalizing, the surfaces were treated with a 0.2% HA HW solution
in presence of NHS and EDC overnight, then washed with milliQ water and incubated
with a 0.75% vancomycin solution in water in presence of NHS and EDC overnight. At
the end of such incubation, the surfaces were washed with milliQ water and dried.
[0061] Releasing step was conducted in PBS at 37°C, with the measurement of the amount released
at 4 hours, 24 hours, 4 days, 12 days and 21 days through HPLC. All the times revealed
that the surfaces modified with collagen release a greater amount of antibiotic with
respect to those functionalised with PEI. The chart in figure 4 shows the cumulative
curve of the release of vancomycin from the differently functionalised surfaces: it
can be observed that, at each time point, the surfaces functionalized with fibrillated
collagen release a greater amount of vancomycin.
[0062] This example indicates that, for surprising and not entirely clear reasons, the surface
functionalization step, in particular the molecular species used for the surface functionalization,
influences the total amount of vancomycin "captured" from the surface coated with
hyaluronic acid and/or the amount of vancomycin that the surface coated with hyaluronic
acid is capable of releasing. Going beyond the importance of this observation from
an applicative point of view, the result confirms that the observed phenomenon is
not a general effect of the action of EDC-NHS on the vancomycin in solution, but it
is bonded in a surprising and unexpected manner to the molecular structure of the
surface.
Example 7 - Verifying the improved properties of stimulation of the osteogenic behaviour
of mesenchymal cells
[0063] In order to evaluate the response of the mesenchymal cells to the coating process
and thus verifying whether the latter can also have effects on osteogenesis, mesenchymal
cells were cultured on the following surfaces, provided on plates with micro-wells
for cellular cultures:
- plastic for cellular cultures (control)
- plastic for cultures functionalised with PEI and subsequent covalent bond of hyaluronic
acid 800kDa (HA)
- plastic for cultures functionalised with PEI and subsequent covalent bond of hyaluronic
acid 800kDa, followed by the formation of HA-Vancomycin complexes as described in
the previous example(HA-VXL)
[0064] Mesenchymal cells from human bone marrow were acquired from Lonza Milano srl in undifferentiated
form. As known, according to external stimuli, these cells can differentiate along
some different paths, including the osteogenic one. The cells were cultured in an
osteogenic medium and the expression of some genes responsible for the formation of
bone tissue thereof was evaluated through Real Time Polymerase Chain Reaction (RT-PCR)
analysis. The data are reported in the following table, where "=" means expression
equivalent to that of the control, "+" expression up to 5 times greater than that
of the control, "++" expression greater than 5 times that of the control.
[0065] The following results were obtained, after 10 days of culture:
Gene |
HA |
HA-VXL |
Alkaline phosphatase |
= |
+ |
RunX2 |
+ |
+ |
Osteocalcin |
+ |
+ |
Bone Sialoprotein |
++ |
++ |
Bone Morphogenetic protein -2 (BMP-2) |
++ |
++ |
[0066] These data confirm that coating with HA stimulates the expression of genes linked
to the formation of bone tissue, confirming the in vivo data cited in literature regarding
the effect of the covalent bond of surface layers of HA on osteointegration (
Morra et al, Covalently-Linked Hyaluronan Promotes Bone Formation around Ti Implants
in a Rabbit Model, published on the Journal of Orthopedic Research, 27:657-663, 2009). In particular, both the RunX2 transcription factor, which controls the cellular
differentiation, and in particular the BMP-2 protein and the BSP (Bone Sialoprotein)
are markedly over-expressed on HA and HA-VXL with respect to the control, and they
indicate an extremely significant osteogenic process. The presence of the complexes
with vancomycin does not substantially alter the advantages of HA (on the contrary,
another very important gene, alkaline phosphatase, appears more expressed on HA-VXL
with respect to HA), confirming that also this type of surface is pro-osteogenic.
This is a very important property, which combined with the peculiar release of the
antibiotic, is at the base of the generation and design of multifunctional devices.
Example 8 - Verification of the antibacterial efficiency of implant screws obtained
according to the present process
[0067] Inhibition areola tests were conducted in
Staphylococcus epidermidis cultures with the aim of proving the antibacterial action of the present invention.
Implants coated with HA and with HA-VXL are provided through the methods described
previously. These implants, alongside the negative control ones (untreated implants),
were then incubated in a semi-solid agar medium together with the bacterial mixture
in a Petri dish at 37°C. The bacteria should proliferate and, in the case of a surface
with antibacterial properties (with release of antibiotic), an inhibition areola,
i.e. an area without bacteria surrounding the implant, should form around the implant.
The results of this experiment confirmed that the developed treatment has antibacterial
activities: actually, around the HA + VXL implants the inhibition areola, which is
not formed around the control implants, is observed. Figure 5 represents a picture
explaining the experiment, with the control implants at the top (only titanium on
the left, Ti coated with HA on the right) and the treated ones at the bottom (HA-VXL,
two replicas), around which the inhibition areola is clearly observable.
[0068] The implants thus treated were subsequently collected and once again submerged into
an agar containing bacteria to verify whether they would maintain their antibacterial
activity: actually, the inhibition areola forms around the treated implants also in
this second incubation, even though having a diameter slightly smaller with respect
to the one formed the first time. The same happened also in the case of a third incubation.
[0069] Thus the surfaces of the HA-VXL implants reveal the greater osteogenic characteristics
of hyaluronic acid, as shown by the example 7, with which the antibacterial properties
revealed by the results exemplified by the photograph indicated in figure 5 are combined.
Thus, this example confirms the multifunctional nature of the surface obtained according
to the present process, and the applicative advantage thereof both with respect to
the conventional device (screw made of titanium) and with respected to the device
coated with HA described in the art.
Example 9 Embodiment of a fracture fixation screw made of titanium, with roughened
surface and coating with hyaluronic acid-vancomycin
[0070] A screw made of titanium degree 5 for fracture fixation is used for the demonstration
of the preparation of an implant device with multifunctional surface, having the following
properties:
- rough surface and ensuing increase of surface area
- bioactive surface through hyaluronic acid bond upon functionalization through adsorption
of fibrillated collagen
- antibacterial surface through the release of the vancomycin present in complexes with
hyaluronic acid bonded to the surface.
[0071] The apical portion (head) of the screw is produced through masking and the screw
is subjected to a sand-blasting process for 40 seconds in a Norblast sandblasting
machine, using titanium oxides as sanding agent. The screw is then subjected to a
treatment process with acids, according to protocols commonly used by this company
to treat dental implants and then subjected to the process in question like in example
7 and 8.
[0072] The roughened surface of the screw has a surface area, and thus a contact surface,
more than 70% greater than that of a conventional screw.
1. Implantable device to be implanted in a human or animal body, wherein at least one
part of the surface of said device is coated with a complex of hyaluronic acid and
glycopeptide antibiotic and wherein said hyaluronic acid has an average molecular
weight comprised between 400.000 and 1.000.000 Da.
2. Implantable device according to claim 1, wherein said device is made of steel, titanium
or alloys thereof with other metals or of plastic material, which are suitable for
applications on human or animal body, or is made of ceramic materials, such as hydroxylapatite
or other forms of calcium phosphates, even in the form of particulate like powders
and granules used as void bone fillers.
3. Implantable device according to claim 1 or 2, wherein said device consists of a dental
implant screw, preferably made of titanium or alloys thereof, possibly of transmucosal
type, or of a screw, preferably made of titanium or alloys thereof, for spinal or
skeletal fixation, or consists of an intervertebral disc, preferably made of titanium,
alloys thereof or cobalt-chromium alloys or made of metal alloys which are commonly
used for these applications.
4. Implantable device according to any one of claims 1 to 3, wherein said glycopeptide
antibiotic is vancomycin.
5. Implantable device according to claims 1 to 4, wherein said hyaluronic acid is immobilized
on the surface of said device.
6. Device according to claim 5, wherein the surface of said device is amined.
7. Device according to claim 5, wherein the surface of said device comprises carboxyl
groups obtained by plasma deposition of acrylic/methacrylic acid.
8. Device according to claim 5, wherein the surface of said device is functionalised
with collagen or extracellular matrix protein molecules, preferably chosen among laminin,
fibronectin and vitronectin, wherein on the surface of said device collagen is adsorbed
in a monomer or fibrillated form.
9. Device according to any one of claims 1 to 8, wherein said hyaluronic acid complex
with a glycopeptide antibiotic can be obtained by treating said hyaluronic acid with
a solution of said glycopeptide antibiotic in the presence of a condensing or cross-linking
agent.
10. Device according to claim 9, wherein said condensing or cross-linking agent is N-hydroxysuccinimide/
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
11. Collagen gel comprising said hyaluronic acid complex with a glycopeptide antibiotic,
as such or lyophilized, wherein said complex of hyaluronic acid with said glycopeptide
antibiotic is as defined in any one of claims 1 to 4.
12. Gel according to claim 11, to be used as filling material around a bone prosthesis
implant.
13. Kit comprising an implantable device as defined in any one of claims 1 to 10 and a
gel as defined in claim 11 or 12.
14. Method for obtaining an implantable device according to any one of claims 1 to 10
or a gel according to claim 11 or 12, comprising the step of forming said hyaluronic
acid complex with said glycopeptide antibiotic by making said hyaluronic acid come
into contact with a solution of said glycopeptide antibiotic, in the presence of a
condensing or cross-linking agent.
15. Method according to claim 14, wherein said condensing or cross-linking agent is N-hydroxysuccinimide/
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.
16. Method according to claim 14 or 15, wherein the surface of said implantable device
is amined in advance through surface plasma deposition of an amine chosen among allylamine,
hexylamine or heptylamine or through solution deposition of polyethyleneimine or acrylic/methacrylic
acid or through treatment employing silanes, such as 3-aminopropyltriethoxysilane.
17. Method according to claim 16, wherein said hyaluronic acid is immobilized onto said
amined surface through condensation with N-hydroxysuccinimide/ 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide, or by treatment of hyaluronic acid with periodate and subsequent reductive
amination.
18. Method according to claim 14 or 15, wherein the surface of said device is functionalized
in advance with collagen.
19. Method according to claim 14 or 15, comprising the formation of a collagen precipitate
in water suspension comprising said hyaluronic acid complex with said glycopeptide
antibiotic, its separation and partial drying to obtain a gel.
20. Antibacterial and osteoinductive implant device according to any one of claims 1 to
10.
1. Implantierbare Vorrichtung zum implantieren in einen menschlichen oder tierischen
Körper, wobei mindesten ein Teil von der Oberfläche der besagten Vorrichtung mit einem
Komplex von Hyaluronsäure und Glycopeptid-Antibiotikum überzogen ist und wobei besagte
Hyaluronsäure ein durchschnittliches Molekulargewicht hat welches zwischen 400.000
und 1.000.000 Da umfasst.
2. Implantierbare Vorrichtung nach Anspruch 1, wobei besagte Vorrichtung aus Stahl, Titan
oder Legierungen davon mit anderen Metallen oder aus Kunststoff-Material gemacht ist,
das geeignet ist für Anwendungen am menschlichen oder tierischen Körper, oder aus
keramischen Materialien gemacht ist, wie Hydroxylapatit oder andere Formen von Kalzium-Phosphaten,
selbst in der Form von Partikeln wie Pulver und Granulaten, die als Knochenersatzmaterialien
verwendet werden.
3. Implantierbare Vorrichtungg nach Anspruch 1 oder 2, wobei besagte Vorrichtung aus
einer dentalen Implantatschraube besteht, bevorzugt gemacht aus Titan oder Legierungen
davon, möglicherweise aus transmukosalen Typ, oder aus einer Schraube, bevorzugt hergestellt
aus Titan oder Legierungen davon, für spinale oder skeletale Fixierung, oder bestehend
aus einer intervertebralen Scheibe, bevorzugt hergestellt aus Titan, Legierungen davon
oder Kobalt-Chrom Legierungen oder hergestellt aus Metalllegierungen, die üblicherweise
für diese Anwendungen verwendet werden.
4. Implantierbare Vorrichtung nach einem der Ansprüche 1 bis 3, wobei besagtes Glykopeptide-Antibiotikum
Vancomycin ist.
5. Implantierbare Vorrichtung nach Anspruch 1 bis 4, wobei besagte Hyaluronsäure auf
der Oberfläche der besagten Vorrichtung immobilisiert ist.
6. Vorrichtung nach Anspruch 5, wobei die Oberfläche der besagten Vorrichtung aminiert
ist.
7. Vorrichtung nach Anspruch 5, wobei die Oberfläche der besagten Vorrichtung Carboxylgruppen
erhalten durch Plasmaabscheidung von Acryl- oder MethacrylSäure umfasst.
8. Vorrichtung nach Anspruch 5, wobei die Oberfläche der besagten Vorrichtung funktionalisiert
ist mit Kollagen oder extrazelluläre Matrix-Protein-Moleküle, bevorzugt ausgewählt
aus Laminin, Fibronektin und Vitronektin, wobei auf der Oberfläche der besagten Vorrichtung
Kollagen adsorbiert ist in einer monomeren oder fibrillierter Form.
9. Vorrichtung nach einem der Ansprüche 1 bis 8, wobei besagter Hyaluron-Säure Komplex
mit einem Glykopeptid-Antibiotikum erhalten werden kann durch Behandlung besagter
Hyaluron-Säure mit einer Lösung aus besagtem Glykopeptid-Antibiotikum in der Anwesenheit
von einem Kondensations- oder Vernetzungsmittel.
10. Vorrichtung nach Anspruch 9, wobei das Kondensations- oder Vernetzungsmittel ein N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)
Carbodiimid ist.
11. Kollagengel, welches besagten Hyaluron-Säure Komplex mit einem Glykopeptid-Antibiotikum,
als solches oder lyophilisiert, umfasst, wobei besagter Komplex aus Hyaluron-Säure
mit besagtem Glykopeptid-Antibiotikum definiert ist wie in einem der Ansprüche 1 bis
4.
12. Gel nach Anspruch 11 zu Verwendung als Füllmaterial um ein Beinprothesenimplantat.
13. Kit umfassend eine implantierbare Vorrichtung definiert wie in einem der Ansprüche
1 bis 10 und ein Gel wie in Anspruch 11 oder 12 definiert.
14. Verfahren zur Erhaltung einer implantierbare Vorrichtung nach einem der Ansprüche
1 bis 10 oder eines Gels nach Anspruch 11 oder 12, umfassend den Schritt der Bildung
des Hyaluron-Säure Komplexes mit dem Glykopeptid-Antibiotikum durch in Kontakt bringen
der Hyaluron-Säure mit einer Lösung aus dem Glykopeptid-Antibiotkum, in der Anwesenheit
eines Kondensations- oder Vernetzungsmittels.
15. Verfahren nach Anspruch 14, wobei besagtes Kondensations- oder Vernetzungsmittel N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)
Carbodiimid ist.
16. Verfahren nach Anspruch 14 oder 15, wobei die Oberfläche der besagten Implantierbare
Vorrichtung im vorab aminiert ist durch Oberflächen-Plasmaabscheidung eines Amines
ausgewählt aus Allylamine, Hexylamine oder Heptylamine oder durch Lösungsabscheidung
von Polyethylenimine, oder Acryl/Methacryl-Säure oder durch Behandlung mit Verwendung
von Silanen wie 3-Aminopropyltriethoxysilan.
17. Verfahren nach Anspruch 16, wobei die Hyaluron-Säure auf der aminierter Oberfläche
immobilisiert ist durch Kondensation mit N-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)
Carbodiimid, oder durch Behandlung von der Hyaluron-Säure mit Periodat und anschließender
reduzierender Aminierung.
18. Verfahren nach Anspruch 14 oder 15, wobei die Oberfläche der besagten Vorrichtung
vorab mit Kollagen funktionalisiert ist.
19. Verfahren nach Anspruch 14 oder 15, welches die Bildung von einem Kollagen-Präzipitat
in Wassersuspension umfasst, welches besagten Hyaluron-Säure Komplex mit besagtem
Glykopeptid-Antibiotikum, dessen Trennung und partielle Trocknung zur Erhaltung eines
Gels umfasst.
20. Antibakterielle und osteoinduktive implantierbare Vorrichtung nach einem der Ansprüche
1 bis 10.
1. Dispositif implantable destiné à être implanté dans le corps d'un humain ou d'un animal,
dans lequel au moins une partie de la surface dudit dispositif est enduite d'un complexe
d'acide hyaluronique et d'antibiotique glycopeptidique et dans lequel ledit acide
hyaluronique a une masse moléculaire moyenne comprise entre 400 000 et 1 000 000 Da.
2. Dispositif implantable selon la revendication 1, dans lequel ledit dispositif est
fabriqué à partir d'acier, de titane ou d'alliages de ceux-ci avec d'autres métaux
ou de matière plastique, qui sont adaptés pour des applications sur le corps d'un
humain ou d'un animal, ou est fabriqué à partir de matières céramiques, telles que
l'hydroxylapatite ou d'autres formes de phosphates de calcium, même sous la forme
de poudres de type particulaire et de granules utilisés comme matières de remplissage
de vides osseux.
3. Dispositif implantable selon la revendication 1 ou 2, dans lequel ledit dispositif
est constitué d'une vis d'implant dentaire, fabriquée de préférence à partir de titane
ou d'alliages de celui-ci, éventuellement du type transmuqueuse, ou d'une vis, fabriquée
de préférence à partir de titane ou d'alliages de celui-ci, pour une fixation spinale
ou squelettique, ou est constitué d'un disque intervertébral, fabriqué de préférence
à partir de titane, d'alliages de celui-ci ou d'alliages de cobalt-chrome ou à partir
d'alliages de métal qui sont communément utilisés pour ces applications.
4. Dispositif implantable selon l'une quelconque des revendications 1 à 3, dans lequel
ledit antibiotique glycopeptidique est de la vancomycine.
5. Dispositif implantable selon les revendications 1 à 4, dans lequel ledit acide hyaluronique
est immobilisé sur la surface dudit dispositif.
6. Dispositif selon la revendication 5, dans lequel la surface dudit dispositif est aminée.
7. Dispositif selon la revendication 5, dans lequel la surface dudit dispositif comprend
des groupes carboxyle obtenus par dépôt par plasma d'acide acrylique/méthacrylique.
8. Dispositif selon la revendication 5, dans lequel la surface dudit dispositif est fonctionnalisée
avec des molécules de collagène ou de protéine de matrice extracellulaire, de préférence
choisies parmi la laminine, la fibronectine et la vitronectine, dans lequel sur la
surface dudit dispositif, du collagène est adsorbé dans une forme monomère ou fibrillaire.
9. Dispositif selon l'une quelconque des revendications 1 à 8, dans lequel ledit complexe
d'acide hyaluronique avec un antibiotique glycopeptidique peut être obtenu en traitant
ledit acide hyaluronique avec une solution dudit antibiotique glycopeptidique en la
présence d'un agent de condensation ou de réticulation.
10. Dispositif selon la revendication 9, dans lequel ledit agent de condensation ou de
réticulation est un N-hydroxysuccinimide/1-éthyl-3-(3-diméthylaminopropyl) carbodiimide.
11. Gel de collagène comprenant ledit complexe d'acide hyaluronique avec un antibiotique
glycopeptidique, tel quel ou lyophilisé, dans lequel ledit complexe d'acide hyaluronique
avec ledit antibiotique glycopeptidique est tel que défini dans l'une quelconque des
revendications 1 à 4.
12. Gel selon la revendication 11, devant être utilisé comme matière de remplissage autour
d'un implant de prothèse osseuse.
13. Kit comprenant un dispositif implantable tel que défini dans l'une quelconque des
revendications 1 à 10 et un gel tel que défini dans la revendication 11 ou 12.
14. Procédé pour obtenir un dispositif implantable selon l'une quelconque des revendications
1 à 10 ou un gel selon la revendication 11 ou 12, comprenant l'étape de formation
dudit complexe d'acide hyaluronique avec ledit antibiotique glycopeptidique en faisant
entrer en contact ledit acide hyaluronique avec une solution dudit antibiotique glycopeptidique,
en la présence d'un agent de condensation ou de réticulation.
15. Procédé selon la revendication 14, dans lequel ledit agent de condensation ou de réticulation
est un N-hydroxysuccinimide/1-éthyl-3-(3-diméthylaminopropyl) carbodiimide.
16. Procédé selon la revendication 14 ou 15, dans lequel la surface dudit dispositif implantable
est aminée à l'avance par un dépôt par plasma superficiel d'une amine choisie parmi
l'alkylamine, l'hexylamine ou l'heptylamine ou par un dépôt par solution de polyéthylèneimine
ou d'acide acrylique/méthacrylique ou par un traitement employant des silanes, telles
que la 3-aminopropyltriéthoxysilane.
17. Procédé selon la revendication 16, dans lequel ledit acide hyaluronique est immobilisé
sur ladite surface aminée par condensation avec du N-hydroxysuccinimide/1-éthyl-3-(3-diméthylaminopropyl)carbodiimide,
ou par traitement de l'acide hyaluronique avec du périodate et une amination réductive
consécutive.
18. Procédé selon la revendication 14 ou 15, dans lequel la surface dudit dispositif est
fonctionnalisée à l'avance avec du collagène.
19. Procédé selon la revendication 14 ou 15, comprenant la formation d'un précipité de
collagène dans une suspension aqueuse comprenant ledit complexe d'acide hyaluronique
avec ledit antibiotique glycopeptidique, sa séparation et son séchage partiel pour
obtenir un gel.
20. Dispositif d'implant antibactérien et ostéo-inducteur selon l'une quelconque des revendications
1 à 10.